SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication

ABSTRACT SARS-CoV-2 is the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19) and poses a significant threat to global health. N protein (NP), which is a major pathogenic protein among betacoronaviruses, binds to the viral RNA genome to allow viral genome packaging and viral particle release. Recent studies showed that NP antagonizes interferon (IFN) induction and mediates phase separation. Using live SARS-CoV-2 viruses, this study provides solid evidence showing that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity. G3BP1 conditional knockout mice (g3bp1fl/fL, Sftpc-Cre) exhibit significantly higher lung viral loads after SARS-CoV-2 infection than wild-type mice. Our findings contribute to the growing body of knowledge regarding the pathogenicity of NPSARS-CoV-2 and provide insight into new therapeutics targeting NPSARS-CoV-2. IMPORTANCE In this study, by in vitro assay and live SARS-CoV-2 virus infection, we provide solid evidence that the SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo. NPSARS-CoV-2 could efficiently suppress G3BP-mediated SG formation and potentiate viral infection by overcoming antiviral innate immunity mediated by G3BP1 in A549 cell lines and G3BP1 conditional knockout mice (g3bp1-cKO) mice, which provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs.


RESULTS
G3BPs interact directly with NPs of betacoronaviruses. To identify proteins associated with SARS-CoV NP, we previously expressed Flag-tagged SARS-CoV NP (NP SARS-CoV ) in 293 cells and immunoprecipitated the cell lysates with anti-Flag antibody (3). The proteins that coprecipitated with NPs were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis after SDS-PAGE. G3BP1 and G3BP2 were both confidently observed in the SARS-CoV NP immunoprecipitates due to the high exclusive spectrum counts, high protein scores and high coverage ( Fig. 1A; see Table S1 in the supplemental material). These results suggested that SARS-CoV NP may interact with G3BPs. To investigate this interaction, lysates of 293T cells cotransfected with NP SARS-CoV -green fluorescent protein (GFP) and Flag-G3BP1 were subjected to anti-Flag immunoprecipitation followed by immunoblotting with anti-GFP antibody. NP SARS-CoV -GFP coprecipitated with Flag-G3BP1 but not the Flag tag. As a control, no NP SARS-CoV -GFP was observed in the IgG immunoprecipitates. These results suggested that NP SARS-CoV may associate with G3BP1 and G3BP2 (Fig. 1B). Next, the associations between G3BPs and NPs of other betacoronaviruses, such as SARS-CoV-2, MERS-CoV, and human coronavirus (HCoV)-OC43, were investigated. The presence of each NP of coronavirus was detected in the anti-Flag or anti-Myc immunoprecipitates of 293T cells ectopically expressing NP and Flag-G3BP1 or Flag-G3BP2 ( Fig. 1C to F). Furthermore, endogenous G3BP1 bound to ectopically expressed NP SARS-CoV-2 in 293T cells (Fig. 1G, left). Similar results were also obtained in reciprocal experiments (Fig. 1G, right). These results collectively demonstrate a broad-spectrum interaction between G3BPs and NPs of betacoronaviruses.
Because COVID-19 remains a worldwide pandemic, we focused on the interaction of G3BPs with SARS-CoV-2 NP. The direct interaction was then validated by far-Western blot and surface plasmon resonance (SPR) analyses. Anti-Flag immunoprecipitates prepared from 293T cells expressing Flag-G3BP1 or Flag-vector were subjected to SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then blotted with soluble His-NP SARS-CoV-2 , and the results showed that His-NP SARS-CoV-2 directly bound G3BP1, but not IgG, in the same lane in vitro (Fig. 1H, left). Consistently, G3BP2 was also observed to interact directly with NP SARS-CoV-2 (Fig. 1H, right). The kinetics of the interaction was further assessed by SPR analysis, and NP SARS-CoV-2 was found to bind with G3BP1 or G3BP2 with equilibrium dissociation constant (K D ) values of 2.69 Â 10 28 M and 1.03 Â 10 27 M, respectively  1I, two left panels). High binding affinity was also observed between NP SARS-CoV and G3BPs (K D values, 2.57 Â 10 28 M and 3.85 Â 10 28 M; Fig. 1I, two right panels). These results supported the assertion that both G3BP1 and G3BP2 interacted strongly and directly with NP SARS-CoV-2 (with a K D of 10 28 to 10 27 M). Notably, NP SARS-CoV-2 showed a stronger (;4-fold) interaction with G3BP1 than G3BP2.
To demonstrate that NP SARS-COV-2 interacts with G3BPs in SARS-CoV-2 infection, the in situ interaction was subjected in the lungs of mice infected with mouse-adapted SARS-CoV-2 (12) through a Duolink proximity ligation assay (PLA) with anti-NP and anti-G3BP1 (or G3BP2) antibodies. In agreement with the findings reported by Zheng (13), NP SARS-CoV-2 is associated with G3BP1 or G3BP2 in situ ( Fig. 2A and B). As a control, little, if any interaction was observed between the S protein of SARS-CoV-2 and G3BP1 (or G3BP2) ( Fig. 2A and B). Notably, the in situ interaction of NP SARS-CoV-2 with G3BPs was observed in the lungs of patients with In situ PLAs were performed using the indicated antibodies to evaluate lung sections from mice (A) and patients (C). The Duolink signals were subsequently visualized in red, and the nuclei were stained with DAPI (49,6-diamidino-2-phenylindole; blue). (B and D) The relative fluorescence intensity per cell shown in panels A and C was analyzed using ImageJ software (at least 20 cells). All quantitative data are shown as the means 6 SDs. **, P , 0.05.

FIG 1 Legend (Continued)
COVID-19 with the S protein of SARS-CoV-2 and G3BP1 (or G3BP2) as a control ( Fig. 2C and D). These results demonstrated that the nucleocapsid protein interacts with G3BPs in SARS-CoV-2 infection. Collectively, these findings demonstrate that G3BPs, including G3BP1 and G3BP2, directly interact with NP SARS-COV-2 .
Domains involved in the G3BPs-NP SARS-CoV-2 association. To delineate the domains involved in the G3BPs-NP SARS-CoV-2 association, anti-Flag immunoprecipitates of full-length or truncated Flag-G3BP1 were incubated with lysates of 293T cells expressing NP SARS-CoV-2 -GFP ( Fig. 3A and B). An analysis of the adsorbates by immunoblotting with anti-GFP antibody demonstrated that the N terminus (amino acids [aa] 1 to 142) of G3BP1, termed the nuclear transport factor 2-like (NTF2L) domain, was responsible for the association with NP SARS-CoV-2  3B). Flag-G3BP1 (or Flag-G3BP2) was then cotransfected with GFP-tagged full-length or truncated NP SARS-CoV-2 into 293T cells. The NP SARS-CoV-2 (aa 181 to 419), but not NP SARS-CoV-2 (aa 1 to 180), was observed to associate with G3BP1, with a significantly weakened binding compared to that of full-length NP SARS-CoV-2 (Fig. 3C). Further, the deletion mutant of the NP confirmed that its 255-364 aa motif was indispensable for G3BP1 association (Fig. 3D). However, the 1-43 aa motif (N-terminal intrinsically disordered region [IDR], N-terminal arginine-rich motif [N-ARM]) was also observed contributing to the association. Furthermore, the association between NP SARS-CoV-2 -Myc and Flag-G3BPs was significantly attenuated by RNase A digestion (Fig. 4A). Consistently, the interactions between truncated G3BP1 and NP or between G3BP1/G3BP2 and truncated NP were not completely disrupted by RNase A digestion ( Fig. 4B and C). These findings suggested that the binding of NP SARS-CoV-2 to G3BPs is partially dependent on the presence of RNA.
SARS-CoV-2 NP suppresses G3BP-mediated SG formation. NP SARS-CoV-2 binds to G3BPs via aa 255 to 364, and this binding event is responsible for the liquid-liquid phase separation of NP (14). Furthermore, the NTF2L domain plays vital roles in G3BP dimerization and recruitment to SGs (15). Earlier studies also suggested that NP suppresses SG formation (13,17). Accordingly, treatment with sodium arsenite (SA) resulted in stronger colocalization between NP and G3BP1, which suggests their association, and SA-induced SG formation in A549 cells ( Fig. 5A) was significantly suppressed in the cells expressing NP SARS-CoV-2 -GFP compared with the cells not expressing NP SARS-CoV-2 in the same field (Fig. 5B, top, and Fig. 5C) or the cells expressing GFP only (Fig. 5B, bottom). Importantly, SA-induced SG assembly was also suppressed by SARS-CoV-2 infection in A549 cells, and SG formation could be markedly rescued by either G3BP1 or G3BP2 overexpression ( Fig. 5D and E). As expected, SARS-CoV-2 NP colocalized significantly with G3BPs in SGs of infected cells. These findings demonstrate that NP SARS-CoV-2 suppresses G3BP-mediated SG formation, possibly by "neutralizing" the G3BPs via protein interactions because the oversupply of G3BP1 or G3BP2 could rescue the effect induced by SARS-CoV-2 infection.
SARS-CoV-2 NP inhibits G3BP1-dependent IFN production and potentiates viral proliferation. G3BP1 potentiates the RIG-I-related pathway via RNF125-mediated K48-linked polyubiquitination in response to viral infection, which results in IRF3 phosphorylation and increased IFN-b transcription (9). In agreement, IRF3 phosphorylation and IFN-b transcription were induced by G3BP1 in the context of poly(IÁC) stimulation ); in addition, the ectopic expression of NP SARS-CoV-2 alone showed fewer but observable effects because the transfection efficiency was only ;30% in A549 cells, whereas the plasmids encoding G3BP1 and NP SARS-CoV-2 were usually equally delivered into the same cell (18). Notably, NP exerted weaker effects in the absence of G3BP1 overexpression (Fig. 6A, lanes 4 and 5, and Fig. 6B), which suggested that NP-G3BP1 or NP polymer-G3BP1 complexes may exert a stronger inhibitory effect on RIG-I activation than NP alone. These results collectively demonstrated that NP SARS-CoV-2 antagonizes G3BP1-mediated IRF3 phosphorylation and IFN-b production.
The effects of G3BP1 on SARS-CoV-2 replication were then evaluated through an analysis of double-stranded RNA (dsRNA), a presumed intermediate of viral replication, in SARS-CoV-2-infected A549 cells. The colocalization of G3BP1 with SARS-CoV-2 dsRNA was observed (Fig. 6C), and dsRNA formation was significantly increased by G3BP1 knockdown ( Fig. 6D and E). The viral genomic RNA levels in culture supernatants were also significantly enhanced by G3BP1 knockdown, as demonstrated by quantitative real-time reverse transcription-PCR (RT-PCR) assays (Fig. 6F). Moreover, G3BP1 ablation in A549 cells (Fig. 6G, right) resulted in elevated viral replication, but this effect was markedly reversed by G3BP1 rescue (Fig. 6G, left). We then investigated the roles of G3BP1 in virus-infected G3BP1 conditional knockout mice (g3bp1-cKO, g3bp1 fl/fl , Sftpc-Cre), in which G3BP1 was specifically knocked out in type II alveolar cells because complete G3BP1 knockout led to neonatal death (19). The lungs of cKO mice showed significantly higher viral loads at 3 days postintranasal infection with a mouse-adapted SARS-CoV-2 virus than those of their wild-type littermates (Fig. 6H). Together, these findings demonstrate that G3BP1 plays important roles in immunity against SARS-CoV-2 infection.

DISCUSSION
Several recent studies have shown that SARS-CoV-2 NP impairs SG formation to promote the viral replication of SARS-CoV-2 via liquid-liquid phase separation (13,16,17,20,21). However, more information about the interactions between NP and G3BPs and more data from live virus-infected cells, mice, or even patients with COVID-19 need to be further investigated.
Compared with the published work, our study presents some novel findings. First, the interaction of NP with G3BPs was assessed in detail via surface plasmon resonance (SPR) analysis, and a strong, direct interaction with a K D at 10 28 to 10 27 M (Fig. 1I) was detected. Second, the results provide the first demonstration of the interaction between NP and G3BPs in the lung tissue of patients with COVID-19 and mouse-adapted SARS-CoV-2-infected mice (Fig. 2). Third, G3BP1 was found to play vital roles in SARS-CoV-2 infection, as shown using g3bp1-cKO (Continued on next page) SARS-Cov-2 NP Inhibit SG Assembly and IFN Production Journal of Virology mice, in which G3BP1 is specifically knocked out in type II alveolar cells (Fig. 6H). Finally, all NPs of all the tested coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-OC43, were found to interact with G3BP ( Fig. 1B to F), which suggested that the suppression of G3BPs-related host innate immunity might be a general or conserved strategy used by coronaviruses. Together, these data provide in-depth evidence showing the mechanism underlying NP-related SARS-CoV-2 pathogenesis through G3BPs. In our study, we revealed the binding site between G3BP1 and NP. Interestingly, aa 1 to 142 of G3BP1 and aa 255 to 364 of NP, involved in G3BP1-NP interaction (Fig. 3B through  D), were both reported to be responsible for homodimerization (14,15). In accordance with the findings that N-ARM was necessary for SARS-CoV-2 N protein aggregation and condensation into SG (22), N-terminal deletion mutants of NP (D1-180 or D1-43) were demonstrated to decrease G3BP1 association significantly (Fig. 3C and D), which suggested that the N-ARM was involved in NP-G3BPs association, possibly by inducing conformation changes. Considering that the receptor-binding domain (RBD, which binds RNA) of G3BPs and the Nterminal domain (NTD) of NP (which binds viral genomic RNA) were not involved in the interaction, the CTD of NP may facilitate the RNA-contributed association because it could also bind RNA (23,24). Moreover, consistent with the RNase A-digested association between NP SARS-CoV-2 -Myc and Flag-G3BPs (Fig. 4A), the interactions between truncated G3BP1 and NP or between G3BP1/G3BP2 and truncated NP were not completely disrupted by RNase A digestion ( Fig. 4B and C). The similar performance of the binding of G3BP mutants in the presence or absence of RNase A indicated that both the existence and spatial conformation of RNA may contribute to the association, but the exact underlying mechanism merits further investigation.
Studies have reported that SARS-CoV-2 infection impairs the IFN response (25,26), and the expression of G3BP1 results in RIG-I activation by restricting the RNF125-mediated K48 ubiquitination and proteasomal degradation of RIG-I (9). In agreement with these findings, we found that G3BP1 could potentiate the phosphorylation of S396 in IRF3 (Fig. 6A), which plays crucial roles in the RIG-I signaling cascade, and this phosphorylation resulted in increased IFN production (27,28). NP reportedly interacts with RIG-I and represses the IFN-b-related pathway (3,5). Our study revealed that NP compromised the inhibition of IRF3 phosphorylation, probably due to the low transfection efficiency in A549 cells. Interestingly, more extensive inhibition was observed in cells ectopically expressing both NP and G3BP1 than in cells expressing only NP ( Fig. 6A and B). These results suggested that NP SARS-CoV-2 overexpression, which results in abundant NP SARS-CoV-2 , particularly in the presence of G3BP1-uncombined NPs, might induce the recruitment of G3BP1-binding proteins during SG formation and thus negatively impact RIG-I activation via the formation of NP-G3BP1 or NP polymer-G3BP1 complexes.
Consequently, the viral dsRNA levels in cells infected with live SARS-CoV-2 were increased by G3BP1-specific siRNA ( Fig. 6D and E). The viral genomic RNA levels were also potentiated by G3BP1 disruption, and the effect was markedly reversed by G3BP1 rescue (Fig. 6G). Moreover, higher viral loads were observed in the lungs of g3bp1-cKO mice than in those of wild-type mice (Fig. 6H). Although the mouse-adapted SARS-CoV-2 virus exhibits one amino acid change in the N protein (D128Y) (29), the mutation, which does not affect the binding

SARS-Cov-2 NP Inhibit SG Assembly and IFN Production
Journal of Virology domain, was unlikely to restrict the G3BP1 interaction. Given these findings, we concluded that G3BP1 is of vital importance for the restriction of SARS-CoV-2 replication. In summary, through an in vitro assay and live SARS-CoV-2 virus infection, this study provides solid evidence that SARS-CoV-2 NP associates with G3BP1 and G3BP2 in vitro and in vivo and that NP SARS-CoV-2 could efficiently suppress G3BPs-mediated SG formation and potentiate viral infection by overcoming G3BP1-mediated antiviral innate immunity, and these findings provide insightful evidence for improving the understanding of highly contagious SARS-CoV-2.

MATERIALS AND METHODS
Ethics statement. This study followed the recommendations detailed in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Center, Academy of Military Medical Sciences, China.
Mice. G3bp1 fl/fL mice and Sftpc-Cre mice of the C57BL/6J background were purchased from GemPharmatech. G3bp1 fl/fL , Sftpc-Cre mice were generated by crossing mice bearing loxP-flanked g3bp1 alleles (g3bp1 fl/fl ) with a transgenic mouse line expressing Cre recombinase under the control of a modified distal promoter of the gene encoding Sftpc (Sftpc-Cre), which was specifically expressed in type II alveolar cells. The genotypes were identified by PCR analysis with mouse tail-tip DNA. Female mice (18 to 22 g, aged 6 to 8 weeks) were used in the experiments.
All mouse experiments were approved by the Animal Center at the Academy of Military Medical Sciences. Cell culture and transfection. 293T cells were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological Industries), 100 IU/mL ampicillin, and 100 mg/mL streptomycin. A549 cells were grown in Ham's F-12K (Kaighn's) medium (Gibco). The cells were transfected with Lipofectamine 2000 (Thermo Fisher) or the TransIT-X2 dynamic delivery system (Mirus).
The A549 cells used for the SARS-CoV-2 infection study were transfected with an ACE2-expressing plasmid prior to infection.
Virus. SARS-CoV-2 and mouse-adapted SARS-CoV-2 have been described previously (12). All experiments involving infectious SARS-CoV-2 were performed in a biosafety level 3 (BSL3) containment laboratory and approved by the Animal Experiment Committee of Laboratory Animal Center, Beijing Institute of Microbiology and Epidemiology.
LC-MS/MS analysis. Anti-Flag antibody immunoprecipitates prepared from Flag-NP SARS-CoV -transfected 293FT cell lysates were resolved via SDS-PAGE, and the protein bands were then excised. After adequate digestion with chymotrypsin, LC-electrospray ionization-MS/MS-resolved peptides were analyzed using a quadrupole time of flight (Q-TOF) 2 MS system (MicroMass), and the data were compared against Swiss-Prot using the Mascot search engine (http://www.matrixscience.com) for peptide identification. The protein score calculated using Mascot was derived from ion scores as a nonprobabilistic basis for the ranking of protein hits. The ion scores were calculated as 210 Â log(P), where P is the probability that the observed match is a random event. Individual ion scores higher than 43 indicate identity or extensive homology (P , 0.05). The exclusive spectrum counts were then analyzed, and the detected protein coverage of the total sample was calculated (see Table S1 in the supplemental material).
In situ PLA. The associations between NP SARS-CoV-2 and G3BP1 or G3BP2 in lung sections from mice and COVID-19 patients were investigated by Duolink in situ PLA (Sigma-Aldrich). In brief, after sections were cut, the samples on glass coverslips were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and fixed in PFA (4%) for 15 min. Antibodies against G3BP1 or G3BP2 and NP SARS-CoV-2 were used according to the manufacturer's instructions for PLA. The protein-protein interactions were visualized as red fluorescent spots under a Zeiss LSM 800 confocal microscope.
Reverse transcription-quantitative RT-PCR. Total cellular RNA was extracted from cells (10 5 cells) using the RNeasy minikit (catalog no. 74126; Qiagen), and total viral RNA was extracted from 2 mL of culture medium supernatant using the QIAamp viral RNA minikit (catalog no. 52906; Qiagen). RT-PCR was performed using the GoTaq 1-Step RT-qPCR system (catalog no. A6020, Promega) or novel coronavirus (2019-nCoV) nucleic acid detection kit (fluorescence RT-PCR). The primers used for gene detection are listed in Table 1, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control for normalization of the RNA levels.
Extraction of viral RNA and quantitative RT-PCR. Tissue homogenates were prepared by the homogenization of whole tissue with an electric homogenizer for 300 s in 500 mL of TRIzol buffer. The homogenates were centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was collected. Viral RNA was extracted using the QIAamp viral RNA minikit according to the manufacturer's protocol. Viral RNA was quantified by quantitative reverse transcription-PCR targeting NP using premix Ex Taq (TaKaRa) with the following primers and probes (Table 1) Protein purification for SPR. The 6ÂHis tag was fused to the N terminus of Flag-G3BP1/G3BP2 in the pcDNA3.1 plasmid. 293T cells were transfected with the indicated plasmids, and 48 h later, the cells were subjected to ultrasonic lysis. We purified 6ÂHis-tagged proteins through Ni-nitrilotriacetic acid (NTA) column affinity chromatography, and they were analyzed by SDS-PAGE and immunoblotting.
Statistical analysis. Patches of SGs or dsRNA were analyzed using ImageJ software, and the boundary-clear patches were counted as individual SGs or dsRNAs. The mean values from at least three independent experiments were analyzed by Student's t test using GraphPad Prism software. Differences with a P value of ,0.05 were considered significant.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.5 MB. X.Z. assisted with some of the experiments. X.B., Y.L., Y.J., and P.L. developed protocols and provided reagents. H.L. wrote the manuscript, and C.C. and X.L. revised the manuscript. All the authors read and approved the final submission.